Affinity Peptidomics: Peptide Selection and Affinity Capture on Hydrogels and Microarrays

Affinity peptidomics relies on the successfully proven approach used widely in mass-spectrometry-based protein analysis, where protein samples are proteolytically digested prior to the analysis. Unlike traditional proteomic analyses, affinity peptidomics employs affinity detection instead of, or in addition to, the mass-spectrometry detection. Affinity peptidomics, therefore, bridges the gap between protein microarrays and mass spectrometry and can be used for the detection, identification and quantification of endogenous or proteolytic peptides on microarrays and by MALDI-MS. Phage display technology is a widely applicable generic molecular display method suitable for studying protein–protein or protein–peptide interactions and the development of recombinant affinity reagents. Phage display complements the affinity peptidomics approach when the latter is used, e.g. to characterise a repertoire of antigenic determinants of polyclonal, monoclonal antibodies or other recombinantly obtained affinity reagents or in studying protein–protein interactions. 3D materials such as membrane-based porous substrates and acrylamide hydrogels provide convenient alternatives and are superior to many 2D surfaces in maintaining protein conformation and minimising non-specific interactions. Hydrogels have been found to be advantageous in performing antibody affinity assays and peptide-binding assays. Here we report a range of peptide selection and peptide-binding assays used for the detection, quantification or validation of peptide targets using array-based techniques and fluorescent or MS detection.